Quantitative determination method for sodium ions

ABSTRACT

PCT No. PCT/JP94/00572 Sec. 371 Date Nov. 29, 1995 Sec. 102(e) Date Nov. 29, 1995 PCT Filed Apr. 6, 1994 PCT Pub. No. WO94/23061 PCT Pub. Date Oct. 13, 1994The present invention relates to a method for quantitatively determining sodium ions in a sample using  beta -galactosidase in an aqueous medium, wherein a  beta -galactosidase reaction is carried out in the presence of a cation which competes with the sodium ion. The method of the present invention is good in accurate determination and reproducibility and enables the simple and quick quantitative determination of sodium ions.

TECHNICAL FIELD

This invention relates to a method for quantitatively determining sodiumions using β-galactosidase in the presence of a cation which competeswith the sodium ion.

BACKGROUND ART

As a method for chemically determining the amount of sodium ions in abiosample, there is known a method utilizing a β-galactosidase reactionwhich increases enzyme activity in proportion to the amount of sodiumions. In this method, Cryptfix™ 221 is used to prevent the enzymereaction from being saturated with excessive sodium ions ClinicalChemistry, 34:2295 (1988)!. There is also disclosed a method whereinlithium ion is used instead of Cryptfix™ 221 in the above-mentioneddetermination method, but no concrete example is disclosed which useslithium ion alone (Japanese Unexamined Patent Publication/PCT No.1-503596) (Dec. 7, 1989).

A method using a bicyclic crown ether such as Cryptfix™ 221 has thefollowing character. Since the dissociation rate of the cryptate issmall, a long time is needed for the prereaction before the start ofmeasurement. Therefore, the operation of the method is not simple. Inaddition, since Cryptfix™ 221 is preincubated with β-galactosidasebecause of the above-mentioned reasons, the stability of β-galactosidaseis damaged by Cryptfix™ 221 and it becomes impossible to quantitativelydetermine sodium ions at a low concentration. Furthermore, the pH of thereaction solution is restricted to the alkaline area. In view of theseproblems, the development of a better method for quantitativedetermination of sodium ions is desired.

DISCLOSURE OF THE INVENTION

The present invention relates to a method for quantitatively determiningsodium ions in a sample using β-galactosidase in an aqueous medium,which method is characterized in that a β-galactosidase reaction iscarried out in the presence of a cation which competes with the sodiumion.

In the present invention, "an aqueous medium" means a liquid containingwater, such as a buffer and physiological saline. As examples of thebuffer, tris(hydroxymethyl)aminomethane-HCl buffer (hereinafter referredto as "Tris-HCl buffer"), phosphate buffer, acetate buffer, succinatebuffer, oxalate buffer, phthalate buffer, borate buffer, glycine buffer,barbital buffer, Good's buffer and the like may be enumerated.

As the sample containing sodium ions, any sample may be used as long asit is miscible with an aqueous medium. Biosamples such as whole bloodand cells that are difficult to measure by the atomic absorptionspectrometry, the flame photometry or the like can be measured by thepresent invention.

As the cation which competes with the sodium ion, an alkali metal ion,such as lithium, potassium, rubidium or cesium ion, or ammonium ion maybe enumerated. These ions may be used independently or in combination.The suitable concentration of the cation which competes with the sodiumion is 130 mM - 0.5M for lithium ion, 20 mM-200 mM for potassium ion,120 mM-500 mM for cesium ion, 20 mM-500 mM for rubidium ion and 50mM-500 mM for ammonium ion.

As a source for lithium or potassium ions, chlorides, nitrates,sulfates, borohydride of these ions and the like may be enumerated. As asource for rubidium or cesium ions, chlorides of these ions and the likemay be enumerated. As a source for ammonium ions, ammonium sulfate,ammonium chloride and the like may be enumerated.

The β-galactosidase in the present invention may be any enzyme as longas it belongs to the enzyme number EC. 3.2.1.23. A β-galactosidasederived from an animal, microorganism or plant, as well as an enzymewhich is obtained by modifying such a β-galactosidase with geneticengineering techniques are included.

As a substrate for β-galactosidase, either synthetic or naturalsubstrates may be used. For example, β-D-galactoside, arylβ-D-galactoside, alkyl β-D-galactoside, 3,6-dihydroxyfluoranβ-D-galactoside, nitrophenyl β-D-pyranoglycoside, nitrophenylβ-D-galactoside, lactinol, lactose, 4-methylumbelliferyl β-D-galactosideand the like may be enumerated. As an activator for β-galactosidase,magnesium sulfate, magnesium chloride, magnesium nitrate or the like isused.

The amount of a substrate for β-galactosidase decreases in the reactionsolution. Changes of the amount of a substrate for β-galactosidase, canbe determined by measuring the decrease of the substrate, such asnitrophenyl β-D-galactoside mentioned above, by the absorptiometry orthe like.

The amount of reaction products can be determined by measuringgalactose, aglycone, 3,6-dihydroxyfluoran, nitrophenol, etc. whichgenerated from the substrate, by the colorimetry absorptiometry,fluorophotometry, oxidation-reduction measuring method, high performanceliquid chromatography or the like. Alternatively, β-galactosidasereaction may be coupled with galactose dehydrogenase or the like and thereduction type coenzyme produced may be quantitatively determined.

Now, preferable embodiments of the method of the present invention forquantitatively determining sodium ions will be described below.

To the above-mentioned buffer solution (50-1000 mM/l solution: pH5.0-9.5), a cation which competes with sodium ion, magnesium ion (1-20mM) and a sample are added. To the resultant solution, 1-12 mM of asubstrate for β-galactosidase or 200-7500 unit/l of β-galactosidase isadded and prereacted at 8°-50° C. for 1 second or more. Next, in thecase where β-galactosidase has been added, 1-12 mM of a substrate forβ-galactosidase is added thereto, and in the case where a substrate forβ-galactosidase has been added, 200-7500 unit/l of β-galactosidase isadded thereto. Then, the resultant solution is reacted at 8°-50° C. for1 second or more. The amount of the substrate for β-galactosidase whichdecreases in the reaction solution is determined as described above, orthe amount of a β-galactosidase reaction product produced in thereaction solution is determined as described above to thereby determinethe amount of the substrate which has been consumed in theβ-galactosidase reaction. In this enzyme reaction, an amount of thesubstrate which is equivalent to the amount of sodium ions in the sampleis consumed. Therefore, the amount of sodium ions can be determinedaccording to the above-mentioned determination method.

In the practice of the method of the present invention, a surfactantsuch as Triton X-100 may be added, if necessary, to prevent theoccurrence of turbidity in the reaction solution. In addition, ifnecessary, bovine serum albumin (BSA) or human serum albumin (HSA) maybe added which reduces the influence of the albumin contained in thesample and also works as a solubilizing agent. Furthermore, proteinssuch as human immunoglobulin and ovalbumin, solubilizing agents such asdimethyl sulfoxide, and antioxidants such as dithiothreitol may beadded, if necessary.

In order to eliminate the interference of di- or trivalent metals and toenhance the effect of the cation which competes with the sodium ion, achelating agent such as ethylenebis(oxyethylenenitrilo)tetraacetic acid(EGTA) and ethylenediaminetetraacetic acid (EDTA) may be used in themethod of the present invention. In addition, in order to improve theaccuracy in the determination and to enhance the effect of the cationwhich competes with the sodium ion, a binder for sodium ions such as amonocyclic crown ether may also be added. As the monocyclic crown ether,18-crown-6, N- 2-(methoxy)ethyl!monoaza-15-crown-5, N-2-(2-methoxyethoxy)ethyl!monoaza-15-crown-5 are typically used. Theconcentration of the monocyclic crown ether is usually 5-100 mM,preferably 10-50 mM. By adding these chelating agent and binder, it ispossible to lower the suitable concentration of the cation competitivewith the sodium ion.

The cation which competes with the sodium ion used in the presentinvention reduces the activity of the sodium ions in the sample to suchan extent that is necessary and sufficient. Therefore, a prereactionbefore the start of measurement does not require much time in thequantitative determination method of the present invention. In addition,it is not necessary to preincubate the cation with β-galactosidase.Accordingly, the stability of β-galactosidase is not damaged and themeasurement can be performed with this enzyme being in a stablecondition. In addition, the applicable pH range of they method of thepresent invention is broader compared to the reaction using Cryptfix™.Therefore, according to the present invention, there is provided a novelmethod for quantitatively determining sodium ions in a biosample whichmethod is quick, simple and good in accurate determination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows calibration curves for sodium ions obtained by using 220 mMor more of lithium ions. In the FIG. 1, marks -◯- and -- represent thecalibration curves for sodium ions when the lithium ion concentrationsare 220 mM and 260 mM, respectively.

FIG. 2 shows calibration curves for sodium ions obtained by using 130 mMor less of lithium ions. In the FIG. 2, marks -◯- and -- represent thecalibration curves for sodium ions when the lithium ion concentrationsare 90 mM and 130 mM, respectively.

FIG. 3 shows calibration curves for sodium ions obtained by usingpotassium ions. In the FIG. 3, marks -◯- and -- represent thecalibration curves for sodium ions when the concentrations ofo-nitrophenyl β-D-pyranoglycoside are 1.5 mM and 3 mM, respectively.

FIG. 4 shows calibration curves for sodium ions obtained by usingammonium ions. In the FIG. 4, marks -◯- and -- represent thecalibration curves for sodium ions when the ammonium ion concentrationsare 50 mM and 100 mM, respectively.

FIG. 5 shows calibration curves for sodium ions obtained by using cesiumions. In the FIG. 5, marks -◯- and -- represent the calibration curvesfor sodium ions when the cesium ion concentrations are 140 mM and 270mM, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below withreference to the following Examples, which should not be construed aslimiting the scope of the present invention.

(EXAMPLE 1) (1) Preparation of Standard Solutions for Determining SodiumIon Calibration Curves

Sodium chloride (Wako Pure Chemicals) was diluted with distilled waterto prepare standard solutions for determining calibration curves for100-180 mM sodium ions contained in reaction solutions.

(2) Quantitative Determination of Sodium Ions

0.05 ml of the standard solution of sodium ion was placed in a testtube. Then, 2.0 ml of 300 mM Tris-HCl buffer (pH 7.4) preheated to 37°C. and containing 1100 unit/l β-galactosidase (Sigma), 3 mMDL-dithiothreitol (Sigma), 11.2 mM magnesium sulfate (Sigma) and 220 mMor 260 mM lithium chloride (Wako Pure Chemicals) was added to the testtube. Then, 1.0 ml of distilled water preheated to 37° C. and containing1.5 mM o-nitrophenyl β-D-pyranoglycoside (Merck) was added thereto,mixed with agitation, and reacted at 37° C. The amount of o-nitrophenolproduced for 1 minute was determined with a spectrophotometer (Hitachi;Model UV3400) based on the absorption intensity of the visible portionat 420 nm. The calibration curves obtained are shown in FIG. 1.

(EXAMPLE 2)

Sodium ions were quantitatively determined in the same manner as inExample 1 except that the lithium chloride concentration was changed to90 mM or 130 mM. The calibration curves obtained are shown in FIG. 2.

Lithium chloride exhibited linearity at a concentration of 130 mM.

(EXAMPLE 3)

Sodium ions were quantitatively determined in the same manner as inExample 1 except that 50 mM potassium chloride was used instead oflithium chloride and that the o-nitrophenyl β-D-pyranoglycosideconcentration was 1.5 mM or 3 mM. The calibration curves obtained areshown in FIG. 3.

(EXAMPLE 4)

Sodium ions were quantitatively determined in the same manner as inExample 1 except that 50 mM or 100 mM ammonium chloride was used insteadof lithium chloride. The calibration curves obtained are shown in FIG.4.

(EXAMPLE 5)

Sodium ions were quantitatively determined in the same manner as inExample 1 except that 140 mM or 270 mM cesium chloride was used insteadof lithium chloride. The calibration curves obtained are shown in FIG.5.

(EXAMPLE 6)

Sodium ions were quantitatively determined in the same manner as inExample 1 except that two serum samples obtained from humans were usedas samples instead of the standard solution for the sodium ioncalibration curves; that 3 mM o-nitrophenyl β-D-pyranoglycoside wasused; and that HSA was added to the reaction solution at a concentrationof 1.5 g/l. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Serum      Measured Value (mM)                                                (Sample No.)                                                                             Example 1  Example 2                                                                              Flame Photometry                               ______________________________________                                        Sample 1   132        134      135                                            Sample 2   148        146      150                                            ______________________________________                                    

As Table 1 clearly shows, the measured values from the method of thepresent invention correspond with the measured values from the flamephotomerry.

(EXAMPLE 7)

A sodium ion standard solution of 150 mM was measured (n=10) in the samemanner as in Example 1 except that 150 mM potassium chloride was usedinstead of lithium chloride and that the o-nitrophenylβ-D-pyranoglycoside concentration was 2 mM. As a result, the coefficientof variation (hereinafter referred to as "CV") in the measured valueswas 1.5-2.8%.

(EXAMPLE 8)

The CV in sodium ion values determined was measured in the same manneras in Example 7 except that the potassium chloride concentration was 75mM and that 19 mM 18-crown-6 was added together with potassium chloride.As a result, the CV was 0.63-0.88%.

(EXAMPLE 9)

A sodium ion standard solution of 150 mM was measured (n=10) in the samemanner as in Example 1 except that the lithium chloride concentrationwas 150 mM; that 50 mM N- 2-(methoxy)ethyl!monoaza-15-crown-5 was addedtogether with lithium chloride; and that the o-nitrophenylβ-D-pyranoglycoside concentration was 2 mM. As a result, the CV in themeasured values was 0.43-0.75%.

Industrial Applicability

According to the present invention, there is provided a method which isgood in accurate determination and reproducibility and enables thesimple and quick quantitative determination of sodium ions.

We claim:
 1. A method for quantitatively determining sodium ion in a sample; comprising the steps of:admixing β-galactosidase and an aqueous medium comprising said sample; reacting said sample with the β-galactosidase in the presence of at least one cation selected from the group consisting of potassium ion, cesium ion and ammonium ion; and correlating the result of said reaction with the quantity of sodium ions in said sample.
 2. The method according to claim 1, wherein said β-galactosidase is reacted in the presence of a monocyclic crown ether.
 3. A method for quantitatively determining sodium ion in a sample, comprising the steps of:admixing β-galactosidase and an aqueous medium comprising said sample; reacting said sample with the β-galactosidase in the presence of lithium ion at a concentration of from 130 mM to 0.5M; and correlating the result of said reaction with the quantity of sodium ions in said sample.
 4. The method according to claim 3, wherein said β-galactosidase is reacted in the presence of a monocyclic crown ether. 